Project Summary Cancer, the uncontrolled division of abnormal cells, is the second leading cause of death in the United States, taking the lives of nearly 600,000 Americans in 2015. 90% of cancer deaths are due to metastasis, a process which involves cancer cells entering into the blood, squeezing through capillaries, and exiting and colonizing distal organs. Understanding the mechanisms underpinning metastasis, including the mechanical forces cells experience, holds great promise for the rational development of cancer therapeutics, and genomic, transcriptional, and proteomic drivers of metastatic spread have been the topic of intensive study. Recent work has demonstrated that the mechanical stress experienced by cancer cells as they migrate through spatial constrictions is sufficient to induce nuclear envelope disruption, and that this rupture leads to double-stranded DNA breaks (DSBs). Separate work has established a causal link between nuclear envelope rupture and large scale genomic rearrangements in micronuclei, but whether mechanically-induced DSBs can cause heritable mutations in the genome and contribute to cancer progression is still unknown. Additionally, shear stresses and physical forces experienced by a cell during metastasis are capable of modulating their phenotype in vitro, hinting at mechanosensitive, and potentially druggable, signaling networks in cancer cells. With this grant, I propose to test whether the mechanical forces encountered by a cancer cell during metastasis induce DNA damage that leads to heritable genomic alterations, assess the impact of these forces on cellular expression profiles and phenotypes, and then identify potential targets for therapeutic intervention in responsive signaling networks. Aim 1 proposes to identify and quantify the DNA damage incurred by cancer cells during mechanical stress. Nuclear envelope disruption and subsequent DNA damage will be measured using live-cell imaging, immunofluorescence microscopy of markers of DNA damage, and single cell gel electrophoresis in several mechanical stress models. Aim 2 will determine the functional significance of mechanical DNA damage and identify affected genomic loci using in vitro models of proliferation, invasiveness, and drug sensitivity, as well as murine models of tumorigenesis and metastatic potential. Aim 3 will dissect the potential transcriptional and proteomic drivers of the phenotypic differences of mechanically stressed cells from the parental population. RNA sequencing and proteomic analyses will be performed to determine the force-dependent transcriptomic and proteomic changes mediating these functional differences. Finally, gene knock-down and overexpression studies will be used to dissect the potential therapeutic benefit of targeting signaling networks mediating response to mechanical stress. By working at the interface of bioengineering, bioinformatics, and cancer genetics, this proposal will help elucidate the role that mechanical forces play in cancer growth and metastasis.